Three-component CRISPR/Cas complex system and uses thereof

Abstract

The invention described herein provides compositions and reagents for assembling a tripartite complex at a specific location of a target DNA. The invention also provides methods for using the complex to, for example, label a specific genomic locus, to regulate the expression of a target gene, or to create a gene regulatory network.

The invention claimed is: 
     
       1. An RNA polynucleotide comprising:
 (1) a DNA-targeting sequence that is complementary to a target polynucleotide sequence; 
 (2) a Cas9-binding sequence; and, 
 (3) at least 5 tandem copies of a PUF domain-Binding Sequence (PBS). 
 
     
     
       2. The polynucleotide of  claim 1 , wherein the DNA-targeting sequence is complementary to the target polynucleotide sequence over about 12-22 nucleotides. 
     
     
       3. The polynucleotide of  claim 1 , wherein the DNA-targeting sequence has a 5′ end nucleotide, and the 5′ end nucleotide is G. 
     
     
       4. The polynucleotide of  claim 1 , further comprising a linker sequence linking the DNA-targeting sequence to the Cas9-binding sequence. 
     
     
       5. The polynucleotide of  claim 1 , wherein the Cas9-binding sequence
 (1) forms a hairpin structure, or 
 (2) is about 37-47 nt or about 42 nt. 
 
     
     
       6. The polynucleotide of  claim 1 , wherein each of the multiple at least 5 tandem copies of the PBS has about 8 nucleotides. 
     
     
       7. The polynucleotide of  claim 1 , comprising
 a PBS of the sequence 5′-UGUAUGUA-3′ that can be bound by the PUF domain PUF(3-2), or 
 a PBS of the sequence 5′-UUGAUAUA-3′ that can be bound by the PUF domain PUF(6-2/7-2). 
 
     
     
       8. The polynucleotide of  claim 1 , comprising 5-35 tandem copies of identical PUF domain-Binding Sequences. 
     
     
       9. The polynucleotide of  claim 1 , comprising 5-35 tandem copies of different PUF domain-Binding Sequences. 
     
     
       10. The polynucleotide of  claim 1 , comprising 5-10 tandem copies of identical PUF domain-Binding Sequences. 
     
     
       11. The polynucleotide of  claim 1 , comprising 5-10 tandem copies of different PUF domain-Binding Sequences. 
     
     
       12. The polynucleotide of  claim 1 , wherein the tandem copies of the PBS are separated from each other by a spacer sequence. 
     
     
       13. A vector comprising the polynucleotide of  claim 1 . 
     
     
       14. A plurality of vectors of  claim 13 , wherein the polynucleotides of the plurality of vectors have a different DNA-targeting sequence, Cas9-binding sequence, and/or PBS copy number or identity, relative to each other. 
     
     
       15. A complex comprising the polynucleotide of  claim 1  and a Cas9 protein. 
     
     
       16. The complex of  claim 15 , further comprising a PUF domain bound to a PBS of the RNA polynucleotide. 
     
     
       17. The complex of  claim 15 , wherein the Cas9 protein is a nuclease-deficient dCas9 protein. 
     
     
       18. A host cell comprising the vector of  claim 13 .